The intracellular sorting of pro-neuropeptides, prohormones and neurotrophins to the regulated secretory pathway (RSP) is essential for processing, storage and release of active proteins and peptides in the neuroendocrine cell. The enzymes that accomplish the processing are specialized proteins that are required to be trafficked properly in order for them to function as appropriate enzymes in the correct compartment. We investigated the mechanism by which the enzyme, prohormone convertase 1 (PC1), was trafficked to the RSP. We found that a sequence within the C-terminal domain of PC1 (aa618-638) that conferred a transmembrane property on PC1 and lipid raft association was required for its correct trafficking to the RSP. We conclude that this type of membrane association of endoproteases maybe necessary for their correct cellular trafficking in endocrine cells.[unreadable] In addition to studying the trafficking of the enzymes that generate the peptide hormones, we have continued to investigate the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), proinsulin and brain derived neurotrophic factor (BDNF) to the RSP. Such studies will lead to the better understanding of diseases related to defects in hormone and neuropeptide targeting such as; obesity, diabetes, memory and learning. Previously we have shown that Carboxypeptidase E, an enzyme in the RSP, acts a binding protein to facilitate the sorting of prohormones such as POMC into the granules of the RSP. Using a CPE knockout (KO) mouse model, we showed that 50% of newly synthesized POMC in primary cultures of the pituitary anterior lobe cells was degraded and suggests that in the absence of efficient sorting to the granules of the RSP due to the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP as evidenced by its ability to be secreted in a stimulated manner. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII) which has been shown to bind POMC in precipitation assays and indeed analysis of pituitary extracts show that the full length 65 kDa form, which was the form shown to bind POMC, is increased in the KO pituitary suggesting that this increase may facilitate sorting of the residual POMC into the RSP.[unreadable] The mechanism of regulating the formation of large dense-core granules (LDCGs) into which the prohomones are sorted within the RSP has been another major focus of our research. We previously showed that chromogranin A (CgA) plays a critical role in the control of LDCG formation and that the mechanism was through the control of granule protein degradation within the Golgi by regulating the levels of a protease inhibitor. We have identified the inhibitor as protease nexin-1 (PN-1) that was transcriptionally activated by CgA. Furthermore, we found that a 3 kDa C-terminal proteolytic fragment of CgA was able to enhance PN-1 transcription and granule biogenesis in 6T3 cells, an endocrine cell line that normally lacks LDCGs. We named this peptide serpinin to indicate its function as an inducer of the serine protease inhibitor, PN-1. Using an antibody against serpinin, a 3 kDa serpinin-like peptide was detected in the medium after high K+ stimulation of AtT-20 cells. We hypothesize that serpinin binds to a cognate receptor on the plasma membrane to cause signaling to the nucleus to enhance PN-1 mRNA transcription. Thus we have uncovered a novel mechanism whereby serpinin, a CgA-derived peptide secreted into the medium along with POMC-derived hormones upon stimulation of pituitary corticotrophs, signals replenishment of LDCGs by transcriptionally activating a protease inhibitor, PN-1, which then stabilizes granule proteins at the TGN to increase LDCG biogenesis. Interestingly, in addition to PN-1, we found that the water channel protein, Aquaporin 1 (AQP1) was also up-regulated in its expression by CgA. Indeed, we have now demonstrated that AQP1 is present in LDCGs of endocrine tissues and appears to play an integral role in the formation and/or function of the RSP. When down-regulated in its expression in AtT20 cells by si-RNA, the number of LDCGs decreased by 66% and in pulse-chase studies there was a defect in regulated secretion of the endogenous ACTH hormone, as well as increased degradation of newly synthesized granule proteins such as POMC and CPE at the Golgi apparatus. This suggests that AQP1 is necessary for maintaining hormone secretion and granule biogenesis in endocrine cells.[unreadable] [unreadable] Post-Golgi transport of hormone and BDNF vesicles for activity-dependent secretion is important in mediating endocrine function and synaptic plasticity. We have investigated the role of the CPE cytoplasmic tail in vesicle movement. We showed that overexpression of the CPE cytoplasmic tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of an endocrine cell line, AtT20; and hippocampal neurons. In live primary pituitary and AtT20 cell images, overexpression of the CPE tail in the cytoplasm inhibited the movement of POMC/CPE-containing vesicles to the processes. S-tagged CPE tail pulled down endogenous microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from cytosol of AtT20 and brain cells. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. Thus this study has uncovered a novel mechanism whereby the vesicular CPE cytoplasmic tail plays a mandatory role in anchoring regulated secretory pathway POMC/ACTH and BDNF vesicles to microtubule-based motors for transport and activity-dependent secretion in endocrine cells and neurons. We recently demonstrated that CPE is not only associated with large dense core vesicles, but also with synaptic vesicles suggesting that the CPE-tail may also be involved in transport of synaptic vesicles.[unreadable] [unreadable] We have begun to study the role of CPE in the nervous system. The CPE KO mice are very obese and we showed that the animals lacked the anorexigenic neuropeptide, CART in the hypothalamus. These animals over-eat, thus providing further evidence linking decrease of this neuropeptide to the cause of obesity. We also demonstrated deficiencies in several behavioral assays including the Morris water maze and object preference tests indicating a problem with learning and memory in CPE KO mice. Indeed electrophysiological measurements have demonstrated a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. The major reason for this defect appears to be because of the complete lack of the CA3 region of the hippocampus, a region susceptible to seizures in epileptic patients. Understanding the reasons for the specific loss of these neurons in the CPE KO animals will provide new and exciting data on CPE-dependant survival factors that help prevent cell death in this and possibly other regions of the brain.